PROJECT SUMMARY/ABSTRACT Hydrogen sulfide (H2S) remains a chemical hazard in the gas and farming industry. It is easy to manufacture from common chemicals and thus represents a potential threat for the civilian population. It is also employed as a method of suicide, for which incidence has recently increased in the US. H2S exerts its toxicity through its high affinity with various metallo-proteins (cytochrome C oxidase) and its interactions with the cysteine residues of proteins. The latter has been recently shown to alter ion channels in cardiomyocytes and neurons. Indeed, during severe H2S intoxication, a reduction in cardiac contractility, associated with a coma, develops within minutes or even seconds leading to death by complete electro- mechanical dissociation of the heart. If the level of intoxication is milder, a rapid and spontaneous recovery of the coma occurs as soon as the exposure stops. However, in many instances, a cardiogenic shock will persist along with a risk of developing debilitating motor or cognitive deficits. One of the major challenges impeding our effort to offer an effective treatment against H2S intoxication after exposure is that the pool of free/soluble H2S almost immediately disappears from the body, preventing agents trapping free H2S (cobalt or ferric compounds) to play their protective role. We found that methylene blue (MB) appears to overcome this challenge: MB drastically decreases the immediate mortality of H2S intoxication-induced cardiogenic shock and improves the long-term neurological outcome. MB appears to counteract the consequences of H2S intoxication related to the persistent pool of protein-bound H2S. The objective of our proposal is twofold. First, we intend to extend our previous findings on the efficacy of MB against H2S intoxication in two animal models (a large mammal -sheep- and unsedated rats), an essential prerequisite under the Animal Rule, in conditions (inhaled H2S) more faithful to human intoxication. The effects of MB on the short and long term outcomes will be established at two different doses administered up to 30 minutes after the end of intoxication. Second, we will continue to explore the mechanisms of action of MB using isolated contracting cardiomyocytes (studies performed by the group of Dr. Joseph Cheung, Temple University), and cortical neurons from acute brain slices in rats (studies performed by the group of Dr. Brady Maher, Lieber Institute for Brain Development, Johns Hopkins University). Since MB has been used for decades for treating methemoglobinemia (1-2 mg/kg iv) in humans, our ultimate goal is to reposition methylene blue, a drug already on the WHO's list of essential medications, as a key treatment of H2S intoxication.